RU2723590C2 - Lyophilized pharmaceutical compositions - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: method for producing a lyophilized pharmaceutical composition containing a compound of formula (I):or a pharmaceutically acceptable salt thereof, involves dissolving the compound in a solvent, containing dimethylsulphoxide and optionally one or more co-solvents for forming a solution, and then removing the solvent and any co-solvents by a lyophilic drying method, wherein the first stage of freezing is carried out, whereat the solution is frozen, reducing its temperature to −20 °C; first heating stage, at which temperature of frozen solution is induced to temperature in range from −15 °C to 5 °C, where solution is stored frozen; a second freezing step, at which solution temperature is reduced to temperature, not more than −20 °C; primary drying stage, including a sublimation step, where DMSO is removed from solution in frozen state by sublimation at low pressure to obtain partially dried product; and a secondary drying stage, on which DMSO is removed by evaporation of partially dried product in unfrozen state at low pressure to obtain lyophilized product. Invention also relates to lyophilised pharmaceutical compositions and use thereof in medicine, particularly for treating a malignant tumor.EFFECT: disclosed method of producing a lyophilized pharmaceutical composition containing a compound of formula (I), more efficient purification of the obtained lyophilised product from the solvent, shorter drying time, as well as shorter time required to obtain a lyophilized product solution.15 cl, 1 dwg, 6 tbl, 4 ex

Description

CROSS REFERENCE

This application claims the priority of provisional application US No. 62/188025, filed July 2, 2015, incorporated herein by reference in full.

BACKGROUND OF THE INVENTION

DNA methylation is a post-replicative chemical modification of DNA. Different malignant tumors can be separated according to their violated DNA methylation profiles (the degree of general or specific DNA methylation), and hypermethylation of specific genes can be associated with a prognosis for cancer of the stomach, lung, esophagus, pancreas and colon. DNA methylation patterns can also be used to predict response to therapy or resistance to therapy with glioma and melanoma. Azacitidine and decitabine are two FDA approved hypomethylating agents that exert their therapeutic effect by inhibiting DNA methylation levels.

Dinucleotide compounds obtained from decitabine for the development of therapy for similar indications were described in US patent No. 7700567 and its equivalent WO2007041071. Drug compositions containing dinucleotide compounds of the type described in WO2007041071 are described in WO2013033176. The contents of each of US7700567, WO2007041071 and WO2013033176 in full are incorporated by reference.

Lyophilization, often referred to as freeze drying, is a dehydration method in which a substrate containing a solvent is frozen and then placed in a vacuum so that the solvent is removed by sublimation, i.e. direct transition from a solid frozen state to a gaseous state.

TURNING ON THE LINK

Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety, as if they were incorporated by reference individually.

SUMMARY OF THE INVENTION

In some embodiments, the invention relates to a method for producing a lyophilized pharmaceutical composition, a method comprising dissolving a compound of formula (1):

or a pharmaceutically acceptable salt thereof in a solvent containing dimethyl sulfoxide (DMSO) to form a solution, where the solvent is then removed by freeze drying to obtain a freeze dried product, where the freeze drying method comprises: (i) a first freezing step in which the solution is frozen, lowering its temperature to a temperature of not more than approximately -20 ° C; (ii) a first heating step in which the temperature of the frozen solution is induced to a temperature in the range of about -15 ° C to about 5 ° C, where the temperature in the range of about -15 ° C to about 5 ° C keeps the solution frozen; (iii) a second freezing step in which the temperature of the solution is lowered to a temperature not greater than about −20 ° C; (iv) a primary drying step, wherein the primary drying step comprises a sublimation step in which DMSO is removed from the solution in a frozen state by sublimation under reduced pressure to obtain a partially dried product; and (v) a secondary drying step in which DMSO is removed by evaporation from a partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product.

In some embodiments, the invention relates to a pharmaceutical composition prepared by a method comprising the steps of: dissolving a compound of formula (1):

(1),

(1),

or a pharmaceutically acceptable salt thereof in a solvent containing dimethyl sulfoxide (DMSO) to form a solution, where the solvent is then removed by freeze drying to obtain a freeze dried product, where the freeze drying method comprises: (i) a first freezing step in which the solution is frozen, lowering its temperature to a temperature of not more than approximately -20 ° C; (ii) a first heating step in which the temperature of the frozen solution is induced to a temperature in the range of about -15 ° C to about 5 ° C, where the temperature in the range of about -15 ° C to about 5 ° C keeps the solution frozen; (iii) a second freezing step in which the temperature of the solution is lowered to a temperature not greater than about −20 ° C; (iv) a primary drying step, wherein the primary drying step comprises a sublimation step in which DMSO is removed from the solution in a frozen state by sublimation under reduced pressure to obtain a partially dried product; and (v) a secondary drying step in which DMSO is removed by evaporation from a partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of the removal of DMSO over time with the course of the lyophilization process. DMSO removal profiles for the four compositions A, B, C and D with different concentrations are shown in Figure 1.

DETAILED DESCRIPTION

This application relates to lyophilized pharmaceutical compositions containing a dinucleotide derived from decitabine, and to methods for producing and using compositions with a nucleotide derived from decitabine.

The present invention relates to improved lyophilized compositions containing a compound of formula (1) or a pharmaceutically acceptable salt thereof, and to a method for producing improved lyophilized pharmaceutical compositions using a freeze drying method. The invention also relates to the use of lyophilized pharmaceutical compositions in medicine and, in particular, to their use for the treatment of malignant tumors.

The present invention provides improved methods for lyophilization of a substrate containing a non-aqueous solvent, for example, DMSO, and a compound of formula (1), or a pharmaceutically acceptable salt thereof. Basically, the methods include two stages of freezing with an intermediate stage of heating (stage annealing) between the two stages of freezing. The methods can be used to remove non-aqueous solvent from the substrate. In certain specific embodiments, the compound in the substrate is a compound of formula (1):

or a pharmaceutically acceptable salt thereof. The present invention also provides lyophilized compositions comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof. In addition, the present invention provides the use of lyophilized pharmaceutical compositions in medicine, in particular for the treatment of malignant tumors.

It has been found that by using two stages of freezing and an intermediate stage of heating (annealing stage) between the two stages of freezing, DMSO can be removed much faster during the subsequent stage of primary drying and that, thus, the length of the stage of secondary drying can be significantly reduced. Without being limited by any theory, it is believed that an intermediate heating step can provide increased porosity, thereby allowing DMSO to more easily evaporate. Thus, significantly more DMSO is removed during the primary drying step.

Freeze drying microscopy (FDM) studies of the formulations showed that even at temperatures below -30 ° C, some residual unfrozen solvent or co-solvent may be present from time to time. As used herein, the term “frozen” thus includes a state in which a solid structure is formed from solvent molecules and / or a co-solvent, but some amount of solvent and / or co-solvent may also be present in the non-frozen, or liquid form.

A method of obtaining a lyophilized pharmaceutical composition

The methods provided herein include a method for preparing a lyophilized pharmaceutical composition containing a compound, for example, a compound of formula (1)) or a pharmaceutically acceptable salt thereof, the method comprising dissolving a compound of formula (1) or a pharmaceutically acceptable salt thereof in a non-aqueous solvent, containing dimethyl sulfoxide and optionally one or more co-solvents to form a solution, and then removing the solvent and any co-solvents by freeze drying to obtain a lyophilized product; where the freeze-drying method includes one or more of the following steps: (i) a first freezing step, in which the solution is frozen, lowering its temperature to a temperature of not more than about -20 ° C; (ii) a first heating step in which the temperature of the frozen solution is induced to a temperature in the range of about -15 ° C to about 5 ° C, where the temperature in the range of about -15 ° C to about 5 ° C keeps the solution frozen; (iii) the second stage of freezing, which occurs after the first stage of heating and in which the temperature of the solution in the frozen state is reduced to a temperature of not more than about -20 ° C; (iv) a primary drying step comprising a sublimation step in which DMSO and one or more co-solvents, if any, are removed from the solution in a frozen state by sublimation under reduced pressure to obtain a partially dried product; and (v) a secondary drying step, in which DMSO and one or more co-solvents, if any, are removed by evaporation from the partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product.

The sequence of the freezing steps and the intermediate heating step (i), (ii), and (iii) can be repeated one or more times before proceeding to the primary drying step (iv). For example, after the first sequence of steps (i), (ii), and (iii) there may be a second sequence of steps (i), (ii), and (iii), and, optionally, a third and fourth sequence of steps (i), ( ii), and (iii) before proceeding to the primary drying step (iv).

The method according to the invention can, for example, reduce the total time for the freeze-drying method by at least a day, and, in some embodiments, up to two days. The method according to the invention may additionally allow more readily reconstitution of the solution than compositions prepared by methods that do not include an intermediate heating step. For example, in some embodiments of the invention, as defined herein, the recovery time of the compositions may be reduced from a time of more than 30 minutes to a time of less than 20 minutes and, in some embodiments, a time of less than 10 minutes.

The lyophilization procedure can be carried out on the device for freeze drying. The freeze-drying apparatus may have a chamber in which freeze-drying containers (for example, freeze-drying bottles) containing a solution can be placed for freeze-drying. The chamber may be connected to a vacuum source (e.g., a vacuum pump) to reduce the pressure inside the chamber. The device may also have components for freezing or heating the contents of the chamber. Before lyophilization, you can get the bulk of a solution of the compound of formula (1) in DMSO and optionally in one or more co-solvents and filter through a filter (e.g. a sterilization filter) before filling in aliquots of lyophilized drying containers (e.g. lyophilized drying bottles) and transfer to the device for freeze drying. Before transferring to the freeze dryer, the containers can be partially covered with a stopper to prevent contamination, however, allowing solvent to be removed during the freeze drying process.

In the following paragraphs, the parameters of the freeze drying method are described in more detail with reference to specific embodiments, sets, subsets, ranges, and individual values for each parameter. For the avoidance of doubt, each embodiment, a plurality, a subset, a range and an individual value determined relative to one of the parameters of a freeze drying method can be combined with each embodiment, a plurality, a subset, a range and an individual value determined relative to any other parameter of a freeze drying method. This application thus describes all combinations of embodiments, sets, subsets, ranges and individual values for each parameter of the freeze drying method.

The temperatures mentioned above and elsewhere in this document with respect to the parameters of the lyophilization method are the temperatures of the shelves in the freeze dryer. Shelves are usually cooled by coolants, the temperatures of which are controlled, and provide a way to determine the temperature of the shelves. Temperature measurements obtained for coolants can be rechecked based on temperatures obtained directly in the lyophilization product in lyophilized drying containers by introducing temperature probes into the selected lyophilized drying containers.

In the first freezing step (i), the solution can be frozen by lowering its temperature to a temperature of not more than about -20 ° C, for example, the temperature can be reduced to no more than about -30 ° C (or not more than about -35 ° C, or not more than approximately -40 ° C, or not more than approximately -41 ° C, or not more than approximately 42 ° C, or not more than approximately -43 ° C, or not greater than approximately -44 ° C). For example, the solution can be frozen by lowering the temperature to a value in the range of from about -40 ° C to about -50 ° C, or from about -42 ° C to about -48 ° C, or from about -43 ° C to about -47 ° C, or from about -44 ° C to about -46 ° C, for example, about -45 ° C.

The first freezing step may include a temperature ramp step, where the temperature drops from the initial temperature (e.g., the external environment) to the target temperature during the first time period, e.g., for a period of up to about 2 hours or up to about 1.5 hours or up to 1 25 hours, for example, approximately 1 hour.

After reaching the target temperature, the frozen solution may be maintained at the target temperature for a second period of time, for example, up to about 3 hours, or up to about 2.5 hours, or up to about 2 hours, for example, about 1.5 hours.

After the first stage of freezing, the solution is subjected to the first stage of heating, in which the temperature of the frozen solution reaches a temperature in the range from -15 ° C to 4 ° C, at which the solution remains in a frozen state. For example, a frozen solution may be warmed to a temperature in the range of from about -5 ° C to about 5 ° C, or from about -3 ° C to about 3 ° C, or from about -2 ° C to about 2 ° C, or from about -1 ° C to about 1 ° C, for example, about 0 ° C.

The first heating step may include a first period of time during which the frozen solution is heated to the target temperature, and a second period of time during which the frozen solution is maintained at the target temperature. For example, the first time period during which the frozen solution is heated to the target temperature can be up to about 2 hours, or up to about 1.75 hours, or up to about 1.5 hours, for example, about 1.3 hours.

After the first stage of heating, the still frozen solution can be subjected to a second stage of freezing, in which the temperature of the solution in the frozen state is reduced to a temperature of not more than about -20 ° C. The temperature can be reduced to no more than about -30 ° C (or no more than about -35 ° C, or no more than about -40 ° C, or no more than about -41 ° C, or not more than about 42 ° C, or not more than about -43 ° C, or not more than about -44 ° C). For example, the temperature of a frozen solution can be reduced to a value in the range of from about -40 ° C to about -50 ° C, or from about -42 ° C to about -48 ° C, or from about -43 ° C to about -47 ° C, or from about -44 ° C to about -46 ° C, for example, about -45 ° C.

After the second freezing step, the frozen solution may be subjected to a primary drying step including a sublimation step in which dimethyl sulfoxide and one or more co-solvents, if any, are removed by sublimation from the solution in a frozen state under reduced pressure to obtain a partially dried product. In the primary drying step, the frozen solution can be heated to facilitate faster sublimation of DMSO, while keeping the solution frozen. For example, a frozen solution can be heated to a temperature in the range of -25 ° C to 0 ° C, or from -22 ° C to -2 ° C, for example, from about -20 ° C to about -5 ° C.

At the stage of primary drying, the frozen solution can be heated in stages. For example, in the first heating step, the temperature can be induced from a temperature of not more than about -30 ° C to a temperature in the range of from about -25 ° C to about -19 ° C (e.g., about -20 ° C), and then maintained at this temperature for a certain supporting period. At this temperature, residual unfrozen solvent and / or co-solvent can be removed by evaporation.

In a second heating step, a temperature can be induced from a temperature in the range of about -25 ° C to about -19 ° C (for example, about -20 ° C), to a temperature in the range of about -10 ° C to about 0 ° C (for example , approximately -5 ° C), and then maintain at this temperature for an additionally defined maintenance period. It should be understood that additional intermediate stages of heating and supporting periods can be added to the first and second stages of heating. As an alternative to heating the frozen solution stepwise, heating can be carried out continuously to achieve the desired target temperature.

At the beginning of the initial drying period, the pressure in the vessel containing the frozen solution can be reduced (usually from atmospheric pressure) to a pressure at which removal of DMSO and optionally other co-solvents can occur. The pressure can be reduced to a pressure lower than 1 mbar, for example, lower than 500 μbar, or less than 100 μbar, or less than 50 μbar. For example, the pressure can be reduced to a pressure of less than 20 μbar, or less than 10 μbar, or from 1 to 10 μbar, or from 4 to 8 μbar, for example, approximately 6 μbar.

The primary drying step may include an initial step of reducing the pressure, at which a constant temperature is maintained, and the pressure is reduced to the target value, followed by heating the frozen solution as defined above. Alternatively, pressure reduction and heating of the frozen solution can be carried out simultaneously.

The primary drying step takes from about 20 to about 60 hours, for example, from about 30 to about 50 hours.

Progress in the primary drying step can be monitored using one or more sensors or sensors present in the lyophilization chamber in the lyophilizer. Sensors or sensors (such as a Pirani pressure gauge) can be used to measure one or more parameters inside the chamber, and thus, certain changes in one or more parameters can indicate the progress of the primary drying and provide methods that determine when sublimation of DMSO and optionally any co-solvents. For example, a sensor or sensor may measure the pressure inside the chamber or the conductivity of the gas in the chamber.

During the sublimation process, the temperature should be below the critical temperature and pressure of the product, so that the product remains frozen. Sublimation is a direct solid-gas phase transition for DMSO. If the conditions exceed the critical temperature and pressure, the product is not frozen and, instead, is a liquid, and DMSO can change from liquid to gas (boil). It is unacceptable that DMSo boil instead of sublimation.

The primary drying step can be carried out at pressures of from about 5 μbar to about 40 μbar. The freezing temperature of the product at these pressures is from about -2 ° C to about -4 ° C. The primary drying step can be carried out at temperatures from about -3 ° C to about -9 ° C. In this temperature range, steam pressure is suitable for rapid sublimation, which leads to a better product. In some embodiments, the pressure is about 20 mbar. In some embodiments, the implementation of the temperature is approximately -6 ° C.

Once the sublimation of DMSO has ceased or has fallen below a specific level, a secondary drying step is initiated. In the secondary drying step, dimethyl sulfoxide and one or more co-solvents, if any, are removed by evaporation from the partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product. Thus, in the secondary drying step, reduced pressure conditions are maintained and the partially dried product is heated to a temperature at which it is no longer frozen. Since the boiling point of DMSO is approximately 189 ° C, the partially dried product can be heated to a temperature of at least about 40 ° C, additionally, as a rule, at least about 45 ° C, for example at least about 50 ° C, or at least about 55 ° C. In some embodiments, the partially dried product is heated to a temperature in the range of about 55 ° C to about 70 ° C, for example, about 65 ° C.

The secondary drying step may include one or more steps of ramping the temperature, in which the partially dried product is heated to the target temperature, each step of ramping the temperature is accompanied by a step of maintaining the temperature. In one embodiment, there is one step of ramping the temperature, followed by one step of maintaining the temperature.

During the secondary drying step, unfrozen solvent molecules are removed to obtain a lyophilized product that contains only low levels of residual DMSO.

It is advisable that the stage of secondary drying was carried out at a temperature of approximately 30 ° C to approximately 65 ° C, for example, approximately 40 ° C.

At the end of the secondary drying step, an inert gas, such as nitrogen, is introduced into the freeze-drying chamber, and containers (e.g. vials) containing the freeze-dried product are completely sealed (for example, using a cork and optionally also lids) under an inert gas.

The lyophilization procedure can be carried out on a solution of the compound of formula (1) or a pharmaceutically acceptable salt thereof in a non-aqueous solvent containing dimethyl sulfoxide and optionally one or more co-solvents.

In some embodiments, water pollution can be avoided at any stage. Not limited to theory, it is believed that the formation of a hydrate, in particular, violates the structure of the product, which does not contribute to easy recovery.

In some embodiments, substantially no co-solvents are present; those. the solvent essentially contains DMSO.

In other embodiments, one or more other non-aqueous co-solvents may be present. When a co-solvent is present, the total volume of the co-solvent can typically be no more than about 25% (v / v) of the total solvent. Additionally, as a rule, the total volume of co-solvent, if present, is not more than about 20%, or not more than about 15%, or not more than about 10%, or not more than about 5% of total volume of solvent. For example, the total volume of the co-solvent may be from about 0% (v / v) to about 5% (v / v) of the total solvent.

A solution intended for lyophilization may contain an amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof in a range of from about 5 mg / ml to about 200 mg / ml, for example, in a range of from about 10 mg / ml to about 150 mg / ml . For example, the solution may contain from about 20 mg / ml to about 120 mg / ml, or from about 22 mg / ml to about 110 mg / ml, or from about 25 mg / ml to about 105 mg / ml, or from about 25 mg / ml to about 100 mg / ml of a compound of formula (1) or a pharmaceutically acceptable salt thereof.

In some embodiments, the implementation of the solution contains from about 40 mg / ml to about 110 mg / ml, or from about 50 mg / ml to about 105 mg / ml of a compound of formula (1) or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the solution contains either 75 mg / ml or 100 mg / ml sodium salt of a compound of formula (1).

Non-limiting examples of pressures that can be used during the process of the invention include about 1 μbar, about 2 μbar, about 3 μbar, about 4 μbar, about 5 μbar, about 6 μbar, about 7 μbar, about 8 μbar, about 9 μbar, about 10 μbar, approximately 15 μbar, approximately 20 μbar, approximately 25 μbar, approximately 30 μbar, approximately 35 μbar, approximately 40 μbar, approximately 45 μbar, approximately 50 μbar, approximately 55 μbar, approximately 60 μbar, approximately 65 μbar, approximately 70 μbar , approximately 80 μbar, approximately 90 μbar, and approximately 100 μbar.

Lyophilized pharmaceutical compositions.

The invention relates to a lyophilized pharmaceutical composition that can be prepared (or which is prepared) by freeze drying, as described herein.

Lyophilized pharmaceutical compositions of the invention are characterized by improved solubility compared to known lyophilized formulations of compounds of formula (1) and their salts. Thus, in another embodiment, the invention relates to a lyophilized pharmaceutical composition comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, which can be prepared by freeze drying as defined herein and which has a dissolution time of not more than 20 minutes at ambient temperature and without mechanical stirring in a non-aqueous solvent containing 65% (vol./about.) propylene glycol, 25% (vol./about.) glycerol and 10% (vol./about.) ethanol.

In some embodiments, the lyophilized pharmaceutical composition has a dissolution time in a non-aqueous solvent of no more than 15 minutes, or no more than 12 minutes.

In specific embodiments, the lyophilized pharmaceutical composition has a dissolution time in a non-aqueous solvent of not more than 10 minutes.

Lyophilized pharmaceutical compositions of the invention are also characterized by reduced levels of residual DMSO solvent. Thus, in another embodiment, the invention relates to a lyophilized pharmaceutical composition comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, which can be obtained by freeze drying, as defined herein, and where in the amount of lyophilized composition obtained from 1 gram solution, the content of residual DMSO is not more than 20 mg or not more than 19 mg. It should be understood that reference to “solution” means a solution of its pharmaceutically acceptable salt in a solvent containing dimethyl sulfoxide and optionally one or more co-solvents. The solvent may be non-aqueous, anhydrous or substantially anhydrous.

In another embodiment, a lyophilized pharmaceutical composition is provided comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, which can be prepared by freeze drying as defined herein, and where any residual DMSO is present in the composition in an amount corresponding to no more than 35 mg per 100 mg equivalent of the free base of the compound of formula (1).

The term “100 mg free base equivalent” refers to the amount by weight of the free base that may be present, or when the compound of formula (1) is in salt form, to the amount by weight of the free base contained in the salt. For example, the amount of residual DMSO per 100 mg of free base equivalent is not more than about 32 mg, or not more than about 31 mg, for example, in the range of about 15 mg to about 35 mg, or from about 20 mg to about 32 mg, or from about 25 mg to about 30 mg.

In another embodiment, a lyophilized pharmaceutical composition is provided comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, which can be prepared by freeze drying as defined herein, and which: (a) has a dissolution time at ambient temperature and without help mechanical stirring in a non-aqueous solvent containing 65% (vol./about.) propylene glycol, 25% (vol./about.) glycerol and 10% (vol./about.) ethanol, not more than 20 minutes (or not more than less than 15, or 12, or 10 minutes); and (b) has a residual DMSO content such that in the amount of lyophilized composition obtained from 1 gram of solution, the residual DMSO content is not more than 20 mg, or not more than 19 mg. The solvent may be non-aqueous, anhydrous or substantially anhydrous.

Lyophilized pharmaceutical compositions of the invention, i.e. compositions that can be obtained by freeze drying, as defined herein, can also be characterized with respect to their improved porosity and increased specific surface area compared with known compositions. The specific surface area can be measured using known methods, such as the Brunauer-Emmett-Teller (BET) adsorption method.

The lyophilized pharmaceutical compositions of the invention can be provided in sealed containers, such as vials (eg, glass vials), optionally containing a protective atmosphere of an inert gas such as nitrogen or argon. Sealed containers can be opened when necessary and contents reconstituted by dissolving in a reconstitute, such as a non-aqueous, anhydrous or substantially anhydrous solvent, before administration to the patient. Examples of solvents in which lyophilized pharmaceutical compositions of the invention can be reconstituted are described in WO2013033176.

In an additional aspect, therefore, the invention relates to a sealed pharmaceutical container containing a lyophilized pharmaceutical composition, as defined herein. The sealed pharmaceutical container may be, for example, a bottle with a stopper and optionally additional components (such as a rim) to hold the stopper in place. The sealed container may optionally contain a protective atmosphere of an inert gas such as nitrogen or argon.

In a specific embodiment, the invention relates to a sealed pharmaceutical container containing a lyophilized pharmaceutical composition as defined herein, wherein the composition contains a compound of formula (1) or a pharmaceutically acceptable salt thereof in an amount corresponding to about 100 mg equivalent of the free base of the compound of formula (1), and where no more than 35 mg of residual DMSO is present in the composition.

Reconstituted formulations prepared from lyophilized pharmaceutical compositions

Lyophilized pharmaceutical compositions of the invention can be reconstituted in solvents, such as non-aqueous, anhydrous or substantially anhydrous solvents, to provide liquid injectable compositions for administration to an individual. Liquid compositions may be intended for administration by subcutaneous injection. Thus, in an additional aspect, the invention relates to a method for producing a liquid injection composition, comprising dissolving a lyophilized pharmaceutical composition, as defined herein, in a solvent, in particular in a non-aqueous solvent.

Non-limiting examples of suitable solvents include propylene glycol, glycerin, ethanol, and any combination of the above. The compositions can be obtained in the form of non-aqueous compounds. The compositions may be anhydrous or substantially anhydrous.

The solvent mixture may contain a percentage of propylene glycol based on either mass or volume. In some embodiments, the percentage of propylene glycol may be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, the percentage of propylene glycol may be not more than 90%, not more than 80%, not more than 70%, not more than 60%, not more than about 90%, not more than about 80%, not more than about 70%, or not more approximately 60%. In some embodiments, the percentage of propylene glycol may be from about 30% to about 90%, from about 45% to about 85%, from about 55% to about 75%, from about 60% to about 70%, from about 30% to approximately 90%, approximately 45% to approximately 85%, approximately 55% to approximately 75%, or approximately 60% to approximately 70%. In some embodiments, the percentage of propylene glycol may be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, approximately 30 %, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, or approximately 90% .

The solvent mixture may contain a percentage of glycerol based on either mass or volume. In some embodiments, the implementation of the percentage of glycerol may be at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, or at least about 30%. In some embodiments, the percentage of glycerol may be no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than about 70%, no more than about 60%, no more than about 50 %, not more than about 40%, or not more than about 30%. In some embodiments, the percentage of glycerol can be from 0% to 50%, from 5% to 45%, from 15% to 35%, from 20% to 30%, from 0% to about 50%, from about 5% to approximately 45%, approximately 15% to approximately 35%, or approximately 20% to approximately 30%. In some embodiments, the percentage of glycerol may be 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, or approximately 50%.

The solvent mixture may contain a percentage of ethanol based on either mass or volume. In some embodiments, the percentage of ethanol may be at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least about 1%, at least about 3%, at least about 5%, at least about 10%, or at least about 15%. In some embodiments, the percentage of ethanol may be no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than about 30%, no more than about 25%, no more than about 20 %, not more than about 15%, or not more than about 10%. In some embodiments, the percentage of ethanol may be from 0% to 30%, from 0% to 25%, from 0% to 20%, from 5% to 15%, from 0% to about 30%, from 0% to about 25%, from 0% to about 20%, or from about 5% to about 15%. In some embodiments, the percentage of ethanol may be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% , 14%, 15%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11% , approximately 12%, approximately 13%, approximately 14%, or approximately 15%.

In some embodiments, the implementation of the solvent or mixture of solvents contains from 45% to 85% propylene glycol, from 5% to 45% glycerol and from 0% to 30% ethanol. In some embodiments, the solvent or solvent mixture comprises from about 45% to about 85% propylene glycol, from about 5% to about 45% glycerol, and from 0% to about 30% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains from 45% to 85% propylene glycol, from 5% to 45% glycerol and from 0% to 30% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains from about 45% to about 85% propylene glycol, from about 5% to about 45% glycerol, and from 0% to about 30% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents is from 45% to 85% propylene glycol, from 5% to 45% glycerol, and from 0% to 30% ethanol. In some embodiments, the solvent or solvent mixture is from about 45% to about 85% propylene glycol, from about 5% to about 45% glycerol, and from 0% to about 30% ethanol.

In some embodiments, the implementation of the solvent or mixture of solvents contains from 55% to 75% propylene glycol, from 15% to 35% glycerol and from 0% to 20% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents contains from about 55% to about 75% propylene glycol, from about 15% to about 35% glycerol, and from 0% to about 20% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains from 55% to 75% propylene glycol, from 15% to 35% glycerol and from 0% to 20% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents includes from about 55% to about 75% propylene glycol, from about 15% to about 35% glycerol, and from 0% to about 20% ethanol. In some embodiments, the solvent or mixture of solvents is from 55% to 75% propylene glycol, from 15% to 35% glycerol, and from 0% to 20% ethanol. In some embodiments, the solvent or solvent mixture is from about 55% to about 75% propylene glycol, from about 15% to about 35% glycerol, and from 0% to about 20% ethanol.

In some embodiments, the implementation of the solvent or mixture of solvents contains from 60% to 70% propylene glycol, from 20% to 30% glycerol, and from 5% to 15% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents contains from about 60% to about 70% propylene glycol, from about 20% to about 30% glycerol, and from about 5% to about 15% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains from 60% to 70% propylene glycol, from 20% to 30% glycerol, and from 5% to 15% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains from about 60% to about 70% propylene glycol, from about 20% to about 30% glycerol, and from about 5% to about 15% ethanol. In some embodiments, the solvent or solvent mixture is 60% to 70% propylene glycol, 20% to 30% glycerol, and 5% to 15% ethanol. In some embodiments, the solvent or solvent mixture is from about 60% to about 70% propylene glycol, from about 20% to about 30% glycerol, and from about 5% to about 15% ethanol.

In some embodiments, the solvent or solvent mixture comprises 65% propylene glycol, 25% glycerol, and 10% ethanol. In some embodiments, the solvent or solvent mixture comprises about 65% propylene glycol, about 25% glycerol, and about 10% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains 65% propylene glycol, 25% glycerol and 10% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents essentially contains approximately 65% propylene glycol, approximately 25% glycerol and approximately 10% ethanol. In some embodiments, the solvent or solvent mixture is 65% propylene glycol, 25% glycerol, and 10% ethanol. In some embodiments, the implementation of the solvent or mixture of solvents is approximately 65% propylene glycol, approximately 25% glycerol and approximately 10% ethanol.

Excipients

The pharmaceutical composition of the invention may be a combination of any of the pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents and / or excipients. The pharmaceutical composition facilitates the administration of the compound to the body. The pharmaceutical compositions can be administered in therapeutically effective amounts in the form of pharmaceutical compositions using various forms and routes of administration, including, for example, intravenous, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, ear, nasal and local introduction.

The pharmaceutical composition may be administered topically or systemically, for example, by injection of the compound directly into the organ, optionally in the form of depot formulations or sustained release formulations. The pharmaceutical compositions may be provided in the form of a quick release formulation, in the form of a sustained release formulation, or in the form of an intermediate release formulation. A quick release form can provide immediate release. A sustained release formulation may provide controlled release or delayed delayed release.

For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharmaceutically acceptable carriers or excipients. Such carriers can be used to formulate tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, thick suspensions and suspensions for oral administration by an individual.

Oral pharmaceutical preparations can be prepared by mixing one or more solid excipients with one or more of the compounds described herein, optionally grinding the resulting mixture, and treating the granule mixture after adding suitable adjuvants, if desired, to obtain tablet cores or dragees. Cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may contain an excipient, such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, varnish solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to tablet or dragee coatings, for example, for identification or to characterize various combinations of doses of the active compound.

Pharmaceutical preparations that can be used orally include hard capsules made from gelatin, as well as soft sealed capsules made from gelatin and a plasticizer such as glycerin or sorbitol. In some embodiments, the implementation of the capsule includes a hard gelatin capsule containing one or more of the pharmaceutical, bovine and vegetable gelatins. Gelatin may be alkaline treated. Hard capsules may contain the active ingredients in admixture with an excipient, such as lactose, binders, such as starches, and / or lubricants, such as talc or magnesium stearate, and stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil, or liquid polyethylene glycols. You can add stabilizers. All compositions for oral administration are available in dosages suitable for such administration.

For buccal or sublingual administration, the compositions may be tablets, lozenges, or gels.

Parenteral injections can be formulated for bolus injection or continuous infusion. The pharmaceutical compositions may be in a form suitable for parenteral injection in the form of a sterile suspension, solution or emulsion in oily or aqueous vehicles, and may contain adjuvants, such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared as oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or substances that increase the solubility of the compounds in order to obtain highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle before use, for example, with sterile pyrogen-free water, 0.9% saline, or 5% dextrose in water.

Active compounds can be administered topically and can be formulated in a number of compositions that can be administered topically, such as solutions, suspensions, lotions, gels, pastes, medical pencils, balms, creams and ointments. Such pharmaceutical compositions may contain solubilizers, stabilizers, tonicity enhancers, buffers and preservatives.

Formulations suitable for transdermal administration of the active compounds may use transdermal delivery vehicles and patches for transdermal delivery, may be lipophilic emulsions or buffered aqueous solutions, dissolved and / or dispersed in a polymer or adhesive. Such patches can be designed for continuous, pulsating delivery of pharmaceutical compounds or delivery as needed. Transdermal delivery can be performed using iontophoretic patches. Additionally, transdermal patches can provide controlled delivery. The absorption rate can be slowed down by using membranes that control the speed or by trapping the compound inside the polymer matrix or gel. Alternatively, absorption enhancers can be used to enhance absorption. An absorption enhancer or carrier may include absorbable pharmaceutically acceptable solvents to facilitate passage through the skin. For example, transdermal devices may be in the form of a dressing containing a support member, a reservoir containing compounds and carriers, a barrier controlling the speed for delivering the compounds to the individual’s skin at a controlled and predetermined speed over an extended period of time, and adhesives to fix the device to the skin or to the eye.

For administration by inhalation, the active compounds may be in the form of an aerosol, aerosol mist or powder. The pharmaceutical compositions are conveniently delivered in the form of an aerosol spray from pressure packs or a nebulizer using a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example, gelatine, for use in an inhaler or insufflator can be formulated with a powder containing a mixture of compounds and a suitable powder base, such as lactose or starch.

The compounds can also be formulated as rectal compositions such as an enema, rectal gels, rectal foams, rectal aerosols, suppositories, gel suppositories, or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone and PEG. In compositions in the form of suppositories, low melting wax such as a mixture of fatty acid glycerides or cocoa butter can be used.

In the practical methods of treatment or use provided herein, therapeutically effective amounts of the compounds described herein are administered in pharmaceutical compositions to an individual with a disease or condition to be treated. In some embodiments, an individual is a mammal, such as a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the individual, the strength of the compounds used, and other factors. The compounds can be used individually or in combination with one or more therapeutic agents as components of mixtures.

Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers containing excipients and excipients that facilitate the processing of the active compounds into preparations that can be used pharmaceutically. The composition can be modified depending on the chosen route of administration. Pharmaceutical compositions containing the compounds described herein can be produced, for example, by mixing, dissolving, granulating, dragee-producing, pulverizing, emulsifying, encapsulating, capturing or compressing.

The pharmaceutical compositions may include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein in the form of a free base or a pharmaceutically acceptable salt. The methods and pharmaceutical compositions described herein include the use of crystalline forms (also known as polymorphs), and active metabolites of these compounds with the same type of activity.

Methods for preparing compositions containing the compounds described herein include formulating the compounds with one or more inert pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include, for example, solutions in which the compound is dissolved, emulsions containing the compound, or a solution containing liposomes, micelles or nanoparticles containing the compound, as described herein. Semi-solid compositions include, for example, gels, suspensions and creams. The compositions may be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or in the form of emulsions. These compositions may also contain minor amounts of non-toxic adjuvants, such as humectants or emulsifiers, pH buffering agents and other pharmaceutically acceptable additives.

Non-limiting examples of dosage forms suitable for use according to the invention include food, food, pellets, lozenges, liquid preparations, elixirs, aerosols, inhalations, sprays, powders, tablets, pills, capsules, gel, gel tablets, nanosuspensions, nanoparticle, microgel, lozenges suppository, aqueous or oil suspensions, ointment, patch, lotion, dentifrice, emulsion, creams, drops, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups, phytoceutics, nutraceuticals and any of them combination.

Non-limiting examples of pharmaceutically acceptable excipients suitable for use according to the invention include granulating agents, binders, lubricants, disintegrating agents, sweeteners, glidants, anti-adhesive agents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavors, coating agents, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobial agents, plant cellulosic material and rolling agents, and any combination thereof.

The composition of the invention may be, for example, an immediate release form or a controlled release composition. An instant release formulation can be formulated to allow the compounds to act quickly. Non-limiting examples of immediate release formulations include instant formulations. The controlled release formulation may be a pharmaceutical formulation that has been adapted so that the release rates and release profiles of the drug can meet physiological and chronotherapeutic requirements, or, alternatively, has been formulated to effectively release the drug at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., synthetic or natural origin), other gelling agents (e.g., gelling dietary fibers), matrix based formulations (e.g., formulations containing polymeric material, at least with at least one active ingredient dispersed in it), granules within the matrix, polymer blends, and granular masses.

The compositions described may optionally include from about 0.001% to about 0.005% by volume of pharmaceutically acceptable preservatives. One non-limiting example of a suitable preservative is benzyl alcohol.

In some, the controlled release formulation is a delayed release form. A delayed release form can be formulated to delay the action of the compound over an extended period of time. A delayed release form can be formulated to delay the release of an effective dose of one or more compounds, for example, for about 4, about 8, about 12, about 16, or about 24 hours.

The controlled release composition may be in the form of a sustained release. A sustained release form can be formulated to maintain, for example, the effects of the compound over an extended period of time. A sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., provide a physiologically effective profile in the blood) for about 4, about 8, about 12, about 16, or about 24 hours.

Non-limiting examples of pharmaceutically acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy , Nineteenth Ed (Easton, Pa .: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, PA 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms , Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

Described methods include the introduction of dinucleotide derivative of decitabine or its pharmaceutically acceptable salt, in combination with a pharmaceutically acceptable carrier. A carrier can be selected in order to minimize any degradation of the active ingredient and to minimize any undesirable side effects in an individual.

The compound of formula (I) or a pharmaceutically acceptable salt thereof, can be conveniently formulated herein into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers. See, for example, Remington's Pharmaceutical Sciences , latest edition, EW Martin Mack Pub. Co., Easton, PA, incorporated herein by reference, which describes typical carriers and conventional methods for the preparation of pharmaceutical compositions that can be used in combination with the preparation of the compounds of the compound described herein. Such pharmaceutical agents may be standard carriers for administering the compositions to humans and human individuals, including solutions such as saline and physiological pH buffered solutions. Other compositions may be administered in accordance with standard procedures. For example, pharmaceutical compositions may also include one or more additional active ingredients, such as antimicrobials, anti-inflammatory drugs, and anesthetics.

Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution may be from about 5 to about 8, and may be from about 7 to about 7.5. Additional carriers include sustained release preparations, such as semipermeable matrices of solid hydrophobic polymers containing a compound of formula (I) or a pharmaceutically acceptable salt thereof, the matrices being in the form of articles with a particular shape, for example , films, liposomes, microparticles or microcapsules.

The described methods relate to the administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof as part of a pharmaceutical composition. In various embodiments, the compositions of the invention may include a liquid containing the active agent in solution, in suspension, or both in solution and suspension. Liquid compositions may include gels. In one embodiment, the liquid composition is aqueous. Alternatively, the composition may take the form of an ointment. In another embodiment, the composition is an in situ aqueous gel composition. In some embodiments, the composition is an in situ aqueous solution forming a gel.

Pharmaceutical formulations may include additional carriers, as well as thickeners, diluents, buffers, preservatives, and surfactants in addition to the compounds described herein. Pharmaceutical formulations may also include one or more additional active ingredients, such as antimicrobials, anti-inflammatory drugs, and anesthetics.

The excipient can play a simple and direct role, being an inert filler, or the excipient, as used herein, can be part of a pH stabilizing system or coating to ensure safe delivery of the ingredients to the stomach.

The compound of formula (I) or a pharmaceutically acceptable salt thereof may also be present in liquids, emulsions, or suspensions for the delivery of active therapeutic agents in aerosol form in the body cavity, such as the nose, pharynx or bronchial tract. The ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to other components in these preparations may vary depending on the dosage form.

Depending on the intended route of administration, the pharmaceutical compositions administered as part of the described methods may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams , gels, or the like, for example, in unit dosage form suitable for a single administration of an exact dose. The compositions may contain, as indicated above, an effective amount of a compound of formula (I) or a pharmaceutically acceptable thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicines, pharmaceuticals, carriers, adjuvants, diluents, etc.

For solid compositions, non-toxic solid carriers include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and pharmaceutical grade magnesium carbonate.

Pharmaceutically Acceptable Salts

The compound of formula (1) and its pharmaceutically acceptable salts can be obtained by the methods described in WO2013033176, and as described below in the examples.

In each of the above aspects and embodiments of the invention, the compound of formula (1) can be used in salt or non-salt form.

Pharmaceutically acceptable salts include, for example, acid addition salts and base addition salts. The acid that is added to the compound to form the acid addition salt may be an organic acid or an inorganic acid. The base that is added to the compound to form the base addition salt may be an organic base or an inorganic base. In some embodiments, the pharmaceutically acceptable salt is a metal salt. In some embodiments, the pharmaceutically acceptable salt is an ammonium salt.

Acid addition salts may occur when acid is added to the compound described herein. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. Non-limiting examples of suitable acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitric acid, sulfuric acid, sulfuric acid, phosphoric acid, nicotinic acid, isonicotinic acid, lactic acid, salicylic acid, 4-aminosuccinic acid, 4-amino acid acid, gentisic acid, gluconic acid, glucuronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, citric acid, citric acid, citric acid, citric acid, citric acid, citric acid, citric acid hydroxymaleic acid, methylmaleic acid, glycolic acid, malic acid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, phenylacetic acid, N-cyclohexyl sulfamic acid, methanesulfonic acid, methanesulfonic acid, methanesulfonic acid, methanesulfonic acid, methanesulfonic acid zolsulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid, 3- phosphoglyceric acid, glucose-6-phosphoric acid and amino acid.

Non-limiting examples of suitable acid addition salts include hydrochloride salt, hydrobromide salt, iodohydrate salt, nitrate salt, nitrite salt, sulfate salt, sulfite salt, phosphate salt, hydrogen phosphate salt, divodorophosphate salt, carbonate salt, bicarbonate salt, nicotinate salt, isonicate lactic acid, salicylate salt, 4-aminosalicylate salt, tartaric acid salt, ascorbate salt, gentisinate salt, gluconate salt, glucuronate salt, sugar salt, formate salt, benzoate salt, glutamate salt, pantothenate salt, acetate salt, butyrate salt , fumaric acid salt, succinic acid salt, citric acid salt, oxalate salt, maleic acid salt, maleic acid hydroxy salt, maleic acid methyl salt, glycolate salt, malic acid salt, cinnamic acid salt, mandelic acid salt, 2-phenoxybenzoate salt, 2- acetoxybenzoate salt, embonate salt, phenylacetate salt, N-cyclohexyl sulfate salt, methanesulfonate salt, ethanesulfonate salt, benzenesulfonate salt, p-toluenesulfonate salt, 2-hydroxyethanesulfonate salt, ethane-1,2-disulfonate salt, 4-methylbenzenesulfonate salt, naphthalene-1-2-c , 5-disulfonate salt, 2-phosphoglycerate salt, 3-phosphoglycerate salt, glucose-6-phosphate salt and amino acid salt.

Metal salts may occur when an inorganic base is added to the compound described herein. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal may be an alkali metal, an alkaline earth metal, a transition metal or a metal of the main group. Non-limiting examples of suitable metals include lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium and zinc.

Non-limiting examples of suitable metal salts include lithium salt, sodium salt, potassium salt, cesium salt, cerium salt, magnesium salt, manganese salt, iron salt, calcium salt, strontium salt, cobalt salt, titanium salt, aluminum salt, copper salt, cadmium salt and zinc salt.

Ammonium salts can occur when ammonia or an organic amine is added to the compound described herein. Non-limiting examples of suitable organic amines include triethylamine, diisopropylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole, pyrazine, pipidine, pipidine N, N'-dibenzylethylenediamine, procaine, chlorprocaine, choline, dicyclohexylamine, and N-methylglucamine.

Non-limiting examples of suitable ammonium salts include a triethylamine salt, a diisopropylamine salt, an ethanolamine salt, a diethanolamine salt, a triethanolamine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N-methylamine piperidine salt, a n-ethylpyridine salt , pyrazoline salt, pipyrazoline salt, imidazole salt, pyrazine salt, pipyrazine salt, ethylene diamine salt, N, N'-dibenzylethylenediamine salt, procaine salt, chloroprocaine salt, choline salt, dicyclohexylamine salt and N-methylglycol.

One specific example of a salt of a compound of formula (1) is the sodium salt.

Therapeutic use

Lyophilized pharmaceutical compositions of the present invention can be used to treat a wide range of diseases that are sensitive to decitabine treatment, including the diseases described herein.

Thus, in other aspects, the invention relates to (i) a lyophilized pharmaceutical composition, as defined herein, for use in medicine; (ii) a lyophilized pharmaceutical composition as defined herein for use in treating a disease as defined herein; (iii) a method for treating a disease as defined herein, the method comprising mixing a lyophilized pharmaceutical composition as defined herein with a pharmaceutically acceptable solvent and administering an effective amount of the mixture to an individual in need thereof; (iv) the use of a lyophilized pharmaceutical composition, as defined herein, for the manufacture of a medicament for the treatment of a disease, as defined herein; (v) a method for treating a malignant tumor in a patient in need thereof, the method comprising reconstituting the lyophilized pharmaceutical composition as defined herein in a pharmaceutically acceptable solvent to prepare a liquid composition containing a compound of formula (1) or a pharmaceutically acceptable salt thereof, and administering to the patient a therapeutically effective amount of a liquid formulation.

Examples of diseases that can be treated using the lyophilized pharmaceutical compositions of the present invention include diseases with unwanted or uncontrolled cell proliferation. Such indications include benign tumors, various types of malignant tumors, such as primary tumors and tumor metastases, restenosis (for example, coronary, carotid and cerebral plaques), hematological disorders, impaired stimulation of endothelial cells (atherosclerosis), damage to body tissues during surgical operations, impaired wound healing, impaired angiogenesis, diseases that cause tissue fibrosis, tunnel syndromes, disorders in tissues that are not highly vascularized, and proliferative responses associated with organ transplantation.

Basically, cells in a benign tumor retain signs of their differentiation and do not divide completely in an uncontrolled manner. A benign tumor is usually localized and non-metastatic. Specific types of benign tumors that can be treated using the present invention include hemangiomas, hepatocellular adenoma, cavernous hemangioma, local nodular hyperplasia, auditory nerve neuromas, neurofibroma, bile duct adenoma, bile duct cystoma, fibroma, lipoma, leiomyeloma, moma, myxomas, nodal regenerative hyperplasia, tarchomas and pyogenic granulomas.

In malignant tumors, cells become undifferentiated, do not respond to body signals that control growth, and multiply in an uncontrolled manner. A malignant tumor is invasive and can spread to distant sites (metastasis). Malignant tumors are mainly divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. A secondary tumor, or metastasis, is a tumor that originated elsewhere in the body, but has now spread to a distant organ. Common pathways for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and paths along tissue planes and spaces of the body (peritoneal fluid, cerebrospinal fluid, etc.)

Examples of malignant tumors are carcinomas, for example, carcinomas of the bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid gland, prostate, gastrointestinal hematopoietic tissue systems or skin tumors such as leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, or Burkitt's lymphoma; tumors of the hematopoietic tissue of myeloid origin, for example, acute and chronic myelogenous leukemia, myelodysplastic syndrome, or promyelocytic leukemia; follicular thyroid cancer; tumors of mesenchymal origin, for example fibrosarcoma or rhabdomyosarcoma; tumors of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; Xeroderma pigmentosa; keratoxanthoma; follicular thyroid cancer; or Kaposi’s sarcoma.

Specific types of malignant tumors or malignant tumors, primary or secondary, that can be treated with this invention include bladder cancer, breast cancer, ovarian cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, and head cancer brain, cancer of the larynx, gall bladder, pancreas, rectum, parathyroid gland, thyroid gland, adrenal gland, nervous tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, ulcer and papillary types , metastatic carcinoma of the skin, osteosarcoma, Ewing's sarcoma, reticular cell sarcoma, myeloma, giant cell tumor, small cell lung cancer, gall bladder stones, islet cell opcholus, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy cell carcinoma hyperplasia, medullary carcinoma, pheochromocytoma, mucous membrane neuromas, intestinal ganglioneuromas, hyperplastic tumor of the corneal nerve, tumor with a marfanoid appearance, Wilma tumor, seminoma, ovarian tumor, uterine leiomyoma, cervical dysplasia and carcinoma in situ, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinosis, , rhabdomyosarcoma, Kaposi’s sarcoma, osteogenic and other sarcomas, malignant hypercalcemia, renal cell carcinoma, true polycythemia, adenocarcinoma, glioblastoma multiforme, leukemia, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and.

In one embodiment, the malignant tumor is selected from myelodysplastic syndrome, acute myelogenous leukemia, ovarian cancer, liver cancer, and colorectal cancer.

Hematologic disorders include impaired blood cell growth, which can lead to dysplastic changes in blood cells and hematologic malignancies, such as various leukemia. Examples of hematologic disorders include, but are not limited to, acute myelogenous leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, and sickle cell anemia.

Treatment of impaired cell proliferation caused by tissue damage during surgery is possible for a number of surgical procedures, including joint surgery, intestinal surgery and a keloid scar. Diseases that produce fibrous tissue include emphysema.

Tunnel syndromes that can be treated with the present invention include carpal tunnel syndrome. An example of a cell proliferation disorder that can be treated with the invention is a bone tumor.

Proliferative responses related to organ transplantation that can be treated with the present invention include proliferative responses involved in potential organ rejection or related complications. Specifically, these proliferative responses can occur during transplants of the heart, lung, liver, kidney, and other organs of the body or organ systems.

Disorders of angiogenesis that can be treated with the present invention include pathological angiogenesis accompanying rheumatoid arthritis, ischemic reperfusion associated with edema and brain damage, cortical ischemia, hyperplasia and hypervascularization of the ovaries (polycystic ovary syndrome), endometriosis, psoriasis, diabetes and other angiogenic eye diseases, such as preterm retinopathy of the premature (retrolental fibroplasia), muscular dystrophy, corneal transplant rejection, neurovascular glaucoma, and Osler-Weber syndrome.

Diseases associated with pathological angiogenesis require vascular growth or stimulate vascular growth. For example, corneal angiogenesis includes three phases: a prevascular latent period, active neovascularization, and vascular maturation and regression. The nature and mechanism of various angiogenic factors, including elements of the inflammatory response, such as white blood cells, platelets, cytokines, and eicosanoids, or unidentified plasma components, remains to be determined.

In some embodiments, lyophilized pharmaceutical compositions of the present invention can be used to treat diseases associated with unwanted or pathological angiogenesis. The method comprises administering to a patient suffering from undesirable or pathological angiogenesis, the pharmaceutical compositions of the present invention alone or in combination with an antitumor agent whose activity as an antitumor agent in vivo is undesirably disturbed by high levels of DNA methylation. The specific doses of these agents needed to inhibit angiogenesis and / or angiogenic diseases may depend on the severity of the condition, route of administration, and related factors as determined by the attending physician. In general, generally accepted and effective daily doses are sufficient to effectively inhibit angiogenesis and / or angiogenic diseases.

Lyophilized pharmaceutical compositions of the present invention can be used to treat a number of diseases associated with unwanted angiogenesis, such as retinal / choroidal neovascularization, corneal neovascularization. Examples of retinal / choroidal neovascularization include, but are not limited to, Best dystrophy, myopia, fossa of the optic nerve, Stargardt’s disease, Paget’s disease, venous obstruction, obstruction of arteries, sickle cell anemia, sarcoid, syphilis, elastic pseudoxanthoma, obstructive sleep disease / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, premature retinopathy, Ilza disease, diabetic retinopathy, macular degeneration, Becket disease, infections caused by retinitis or choroiditis, presumptive histoplasmosis syndrome, parsplanitis, chronic retinal detachment syndrome blood, toxoplasmosis, trauma and complications after a laser, diseases associated with rubeosis (angle neovascularization), and diseases caused by pathological proliferation of fibrovascular or vibrous tissue, including all forms of proliferative vitreoretinopathy. Examples of neovascularization of the cornea include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, prolonged wearing of contact lenses, atopic keratitis, upper limbal keratitis, dry pterygium keratitis, Sjogren's syndrome, redheads, phylenetenulosis, diabetic retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, retinopathy, and retinopathy Moray eels, Terriene marginal degeneration, marginal keratolysis, polyarteritis, Wegener's sarcoidosis, scleritis, peripheral radial keratotomy, neovascular glaucoma and retrolental fibroplasia, syphilis, mycobacterial infections, lipid dystrophy, chemical burns, bacterial infections, infections of the herpes zoster virus, infections associated with protozoa, and Kaposi’s sarcoma.

In some embodiments, the lyophilized pharmaceutical compositions of the present invention can be used to treat chronic inflammatory diseases associated with pathological angiogenesis. The method comprises administering to a patient suffering from a chronic inflammatory disease associated with pathological angiogenesis, the pharmaceutical compositions of the present invention alone or in combination with an antitumor agent whose activity as an antitumor agent in vivo is undesirably disturbed by high levels of DNA methylation. Chronic inflammation depends on the continuous formation of capillary processes to maintain the flow of inflammatory cells. The influx and presence of inflammatory cells produce granulomas and thus support a chronic inflammatory condition. Inhibition of angiogenesis with the pharmaceutical compositions of the present invention can prevent the occurrence of granulomas, thereby alleviating the disease. Examples of chronic inflammatory diseases include, but are not limited to, inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, psoriasis, sarcoidosis, and rheumatoid arthritis.

Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are characterized by chronic inflammation and angiogenesis in various parts of the gastrointestinal tract. For example, Crohn’s disease occurs as a chronic transmural inflammatory disease that most commonly affects the distal ileum and colon, but can also occur in any part of the gastrointestinal tract from the mouth to the anus and perianal region. Crohn’s patients usually have chronic diarrhea associated with abdominal pain, fever, anorexia, weight loss, and bloating. Ulcerative colitis is also a chronic non-specific inflammatory and ulcerative disease that occurs in the mucous layer of the colon, which is characterized by bloody diarrhea. These inflammatory bowel diseases are mainly caused by chronic granulomatous inflammation throughout the gastrointestinal tract, including the germination of new capillaries surrounded by a cylinder of inflammatory cells. Inhibition of angiogenesis with the pharmaceutical compositions of the present invention should prevent the formation of germination and prevent the formation of granulomas. Inflammatory bowel disease also exhibits extraintestinal manifestations, such as skin lesions. Such lesions are characterized by inflammation and angiogenesis and can occur in many areas outside the gastrointestinal tract. Inhibition of angiogenesis using the lyophilized pharmaceutical compositions of the present invention should reduce the influx of inflammatory cells and prevent the formation of lesions.

Sarcoidosis, another chronic inflammatory disease, is characterized as a multisystem granulomatous disorder. Granulomas of this disease can form everywhere in the body, and thus, the symptoms depend on the location of the granulomas and whether the disease is active. Granulomas come from the angiogenic germination of capillaries, providing constant nutrition of inflammatory cells. By using the lyophilized pharmaceutical compositions of the present invention to inhibit angiogenesis, the formation of such granulomas can be inhibited. Psoriasis, also a chronic and recurrent inflammatory disease, is characterized by papules and plaques of various sizes. Treatment using the pharmaceutical formulations of the present invention can reduce the likelihood of new blood vessels forming to maintain characteristic lesions and provide the patient with symptom relief.

Rheumatoid arthritis (RA) is also a chronic inflammatory disease that is characterized by non-specific inflammation of the peripheral joints. It is believed that the blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to the formation of new vascular networks, endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. Factors involved in angiogenesis can make an active contribution to chronic inflammation in rheumatoid arthritis and help maintain it. Treatment with the pharmaceutical formulations of the present invention alone or in combination with other anti-RA agents can reduce the likelihood of new blood vessels forming to maintain chronic inflammation and provide symptomatic relief for a patient with RA.

In some embodiments, lyophilized pharmaceutical compositions of the present invention can be used to treat diseases associated with impaired hemoglobin synthesis. The method comprises administering the pharmaceutical compositions of the present invention to a patient suffering from a disease associated with impaired hemoglobin synthesis. Compositions containing decitabine stimulate the synthesis of fetal hemoglobin, since the mechanism of incorporation into DNA is associated with DNA hypomethylation. Examples of diseases associated with impaired hemoglobin synthesis include, but are not limited to, sickle cell anemia and β-thalassemia.

In some embodiments, lyophilized pharmaceutical compositions of the present invention can be used to control intracellular gene expression. The method includes administering the pharmaceutical compositions of the present invention to a patient suffering from a disease associated with abnormal gene expression levels. DNA methylation is associated with control of gene expression. Specifically, methylation in or adjacent to promoters inhibits transcription, while demethylation restores expression. Examples of possible uses of the described mechanisms as non-limiting examples include therapeutically altered growth inhibition, induction of apoptosis, and cell differentiation.

In some embodiments, the lyophilized pharmaceutical compositions of the invention can be used to treat patients with genetic mutations associated with tumor hypermethylation, such as patients with types of tumors that contain a mutation or deficiency of succinate dehydrogenase (SDH), and include patients with non-KIT mutated gastrointestinal intestinal stromal tumors (GIST).

Facilitating gene activation using the lyophilized pharmaceutical compositions of the present invention may cause cell differentiation for therapeutic purposes. Cellular differentiation is induced through the mechanism of hypomethylation. Examples of morphological and functional differentiation as non-limiting examples include differentiation in the direction of the formation of muscle cells, muscle tubes, erythroid and lymphoid germ cells.

Myelodysplastic syndromes (MDS) are heterogeneous disorders in clonal hematopoietic stem cells associated with the presence of dysplastic changes in one or more hematopoietic shoots, including dysplastic changes in the myeloid, erythroid and megakaryotic series. These changes lead to cytopenia in one or more of the three sprouts. Individuals suffering from MDS usually develop complications associated with anemia, neutropenia (infection), or thrombocytopenia (bleeding). In general, from about 10% to about 70% of individuals with MDS develop acute leukemia. Typical myelodysplastic syndromes include acute myelogenous leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, and chronic myelogenous leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Some hereditary genetic disorders and immunodeficiency conditions are associated with an increased risk of AML. These include abnormalities with DNA stability defects leading to an accidental chromosomal rupture, such as Bloom syndrome, Fanconi anemia, a genealogy with Li-Fraumeni syndrome, ataxia-telangiectasia, and X-linked agammaglobulinemia.

Acute promyelocytic leukemia (AML) is a separate subtype of AML. This subtype is characterized by promyelocytic blasts containing chromosomal translocation 15; 17. This translocation leads to the production of a fusion transcript containing the retinoic acid receptor sequence and the sequence of promyelocytic leukemia.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with various subtypes that demonstrate various clinical features. In ALL, repeated cytogenetic abnormalities have been demonstrated. The most common concomitant cytogenetic abnormality is translocation 9; 22, leading to the development of the Philadelphia chromosome.

In a specific embodiment, the lyophilized pharmaceutical compositions of the present invention can be used to treat MDS, for example, MDS selected from AML, OPML and ALL.

It should be understood that in each of the above therapeutic applications, the lyophilized pharmaceutical compositions of the invention are typically reconstituted in a suitable solvent, as defined herein, before administration to an individual, for example, a mammalian individual, such as a human patient.

Dosage and Administration

Dosages of the lyophilized pharmaceutical compositions of the invention reconstituted or mixed as necessary with a pharmaceutically acceptable solvent or mixture of solvents as defined herein can be administered to an individual by methods known in the art. Non-limiting examples of routes of administration include subcutaneous injection, intravenous injection, and infusion.

The dose of the composition contains an amount that is therapeutically effective for the treatment of the disease. A therapeutically effective amount of a compound of the invention can be expressed as mg of compound per kg body weight of an individual. In some embodiments, the therapeutically effective amount is 1-1000 mg / kg, 1-500 mg / kg, 1-250 mg / kg, 1-100 mg / kg, 1-50 mg / kg, 1-25 mg / kg, or 1-10 mg / kg. In some embodiments, the therapeutically effective amount is 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 25 mg / kg, approximately 50 mg / kg, approximately 75 mg / kg, approximately 100 mg / kg, approximately 150 mg / kg, approximately 200 mg / kg, approximately 250 mg / kg, approximately 300 mg / kg, approximately 400 mg / kg, approximately 500 mg / kg, approximately 600 mg / kg, approximately 700 mg / kg, approximately 800 mg / kg, approximately 900 mg / kg, or approximately 1000 mg / kg.

The compound described herein may be present in the composition in the range of about 1 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg, about 20 mg to approximately 25 mg, approximately 25 mg to approximately 30 mg, approximately 30 mg to approximately 35 mg, approximately 35 mg to approximately 40 mg, approximately 40 mg to approximately 45 mg, approximately 45 mg to approximately 50 mg, approximately 50 mg to approximately 55 mg, approximately 55 mg to approximately 60 mg, approximately 60 mg to approximately 65 mg, approximately 65 mg to approximately 70 mg, approximately 70 mg to approximately 75 mg, approximately 75 mg to about 80 mg, about 80 mg to about 85 mg, about 85 mg to about 90 mg, about 90 mg to p approximately 95 mg, approximately 95 mg to approximately 100 mg, approximately 100 mg to approximately 125 mg, approximately 125 mg to approximately 150 mg, approximately 150 mg to approximately 175 mg, approximately 175 mg to approximately 200 mg, approximately 200 mg to about 225 mg, about 225 mg to about 250 mg, or about 250 mg to about 300 mg.

The compound described herein may be present in the composition in an amount of approximately 1 mg, approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 45 mg, approximately 50 mg, approximately 55 mg, approximately 60 mg, approximately 65 mg, approximately 70 mg, approximately 75 mg, approximately 80 mg, approximately 85 mg, approximately 90 mg, approximately 95 mg, approximately 100 mg, approximately 125 mg approximately 150 mg, approximately 175 mg, approximately 200 mg, approximately 225 mg, approximately 250 mg, or approximately 300 mg.

In some embodiments, a therapeutically effective amount may be administered 1-35 times a week, 1-14 times a week, or 1-7 times a week. In some embodiments, a therapeutically effective amount may be administered 1-10 times per day, 1-5 times per day, 1 time, 2 times, or 3 times per day.

It is believed that the lyophilized pharmaceutical compositions of the invention will be useful individually or in combination therapy with other chemotherapeutic agents or radiation therapy for the prevention or treatment of a number of proliferative diseases or conditions. Examples of such diseases and conditions are listed above.

The lyophilized pharmaceutical compositions of the invention, whether administered alone or in combination with antitumor agents and therapy, such as radiation therapy, are typically administered to an individual who needs such administration, for example, to a human patient or animal patient, preferably a human.

Examples of chemotherapeutic agents that can be co-administered with the lyophilized pharmaceutical compositions of the invention, as defined herein, include, but are not limited to, topoisomerase I inhibitors, other antimetabolites, tubulin targeting agents, a DNA binding agent and topoisomerase II inhibitors, alkylating agents monoclonal antibodies, anti-hormonal agents, signal transduction inhibitors, proteasome inhibitors, DNA methyltransferase inhibitors, cytokines, interferons, interleukins, retinoids, chromatin-targeted therapeutic agents, for example, HDAC or HAT modulators, T-cell activators, including immunomodulatory antibodies, vaccines against malignant tumors, hormones, herbal remedies, biological agents, immunomodulating agents, radiation therapy and other therapeutic or prophylactic agents, for example, agents that reduce or relieve detect some of the side effects associated with chemotherapy, such as anti-emetics and drugs that prevent or reduce the duration of chemotherapy-related neutropenia and prevent complications that result from decreased levels of red blood cells or white blood cells, such as erythropoietin (EPO), granulocyte macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF).

In one embodiment, the lyophilized pharmaceutical compositions of the invention are used in combination with histone deacetylase inhibitors (HDACs) (or they additionally contain them) to further modify gene transcription in a synergistic manner, for example, to restore transcription of genes turned off by histone hypermethylation and acetylation.

HDAC inhibitors include, but are not limited to, the following structural classes: 1) hydroxamic acids; 2) cyclic peptides; 3) benzamides; and 4) short chain fatty acids. Examples of hydroxamic acids and hydroxamic acid derivatives include trichostatin A (TSA), hydroxamic acid suberoyl anilide (SAHA), oxamflatin, suberinic bis-hydroxamic acid (SBHA), m-carboxy-cinnamic acid-bis-hydroxamic acid (CBHA) and pyroxamide. TSA was isolated as an antifungal antibiotic (Tsuji et al (1976) J. Antibiot (Tokyo) 29: 1-6) and was found to be a possible inhibitor of mammalian HDAC (Yoshida et al. (1990) J. Biol. Chem 265: 17174-17179). The discovery that TSA-resistant cell lines have altered HDAC suggests that this enzyme is an important target for TSA. Other hydroxamic acid-based HDAC inhibitors, SAHA, SBHA, and CBHA are synthetic compounds that are capable of inhibiting HDAC at micromolar concentrations or lower in vitro or in vivo . Glick et al. (1999) Cancer Res. 59: 4392-4399. All of these hydroxamic acid-based HDAC inhibitors have the necessary structural feature: a polar hydroxam end linked through a hydrophobic methylene spacer (e.g., 6 carbons in length) to another polar region that is attached to the terminal hydrophobic group (e.g., benzene ring).

The cyclic peptides used as HDAC inhibitors are mainly cyclic tetrapeptides. Examples of cyclic peptides include, but are not limited to, trapoxin A, apicidin, and FR901228. Trapoxin A is a cyclic tetrapeptide that contains a 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) group. Kijima et al. (1993) J. Biol. Chem. 268: 22429-22435. Apicidin is a metabolite of fungi that exhibits potent broad-spectrum activity against protozoa and inhibits HDAC activity in nanomolar concentrations. Darkin-Rattray et al. (1996) Proc. Natl. Acad. Sci. USA 93; 13143-13147. FR901228 is a depsipeptide that is isolated from Chromobacterium violaceum and has been shown to inhibit HDAC activity at micromolar concentrations.

Examples of benzamides as non-limiting examples include MS-27-275. Saito et al. (1990) Proc. Natl. Acad. Sci. USA 96: 4592-4597. Examples of short chain fatty acids include, but are not limited to, butyrates (e.g., butyric acid, arginine butyrate, and phenyl butyrate (PB)). Newmark et al. (1994) Cancer Lett. 78: 1-5; and Carducci et al. (1997) Anticancer Res. 17: 3972-3973. In addition, depudecin, which has been shown to inhibit HDAC at micromolar concentrations (Kwon et al. (1998) Proc. Natl. Acad. Sci. USA. 95: 3356-3361), is also included in the volume of the histone deacetylase inhibitor of the present invention.

In one embodiment, the alkylating agent is used in combination with the lyophilized pharmaceutical compositions of the invention. Examples of alkylating agents include bichloroethylamines (nitrogen mustards, e.g. chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uraciliprit), aziridines (e.g. thiotepa), alkyl alkanesulfonates (e.g. busulfan), nitrosourea urea, e.g. non-classical alkylating agents (altretamine, dacarbazine, and procarbazine), platinum compounds (carboplatin and cisplatin).

In another embodiment, the lyophilized pharmaceutical composition of the invention is used in combination with a platinum compound such as cisplatin or carboplatin.

In another embodiment, the lyophilized pharmaceutical composition of the invention is used in combination with a member of the retinoid superfamily, such as fully transretinol, fully trans-retinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin) and 9-cis retinoic acid.

In a further embodiment, the lyophilized pharmaceutical composition of the invention is used in combination with a hormonal agent such as synthetic estrogen (e.g. diethylstibestrol), anti-estrogen (e.g. tamoxifen, toremifene, fluoxymesterol and raloxifene), antiandrogen (bicalutamide, nilutamide inhibitor, nilutamide), (e.g. aminoglutethimide, anastrozole and tetrazole), ketoconazole, goserelin acetate, leuprodide, megestrol acetate and mifepristone.

In yet another embodiment, the lyophilized pharmaceutical composition of the invention is used in combination with a herbal product such as vinca alkaloid (e.g. vincristine, vinblastine, vindesine, vinzolidine and vinorelbine), camptothecin (20 (S) -camptothecin, 9-nitro-20 (S) -camptothecin, and 9-amino-20 (S) -camptothecin), podophyllotoxin (e.g. etoposide (VP-16) and teniposide (VM-26)), and taxane (e.g. paclitaxel and docetaxel).

In a specific embodiment, the lyophilized pharmaceutical composition of the invention is used in combination with a taxane, such as paclitaxel and docetaxel.

In another embodiment, the lyophilized pharmaceutical compositions of the invention can be used in combination with anthracycline, such as daunorubicin or idarubicin.

In a further embodiment, the lyophilized pharmaceutical composition of the invention is used in combination with a biological agent, such as an immunomodulating protein (e.g., a cytokine), a monoclonal antibody against a tumor antigen, a tumor suppressor gene, or a cancer vaccine.

Examples of interleukins that can be used in combination with the lyophilized pharmaceutical composition of the invention include, but are not limited to, interleukin 2 (IL-2) and interleukin 4 (IL-4), interleukin 12 (IL-12). Examples of interferons that can be used in combination with the lyophilized pharmaceutical composition of the invention include, but are not limited to, interferon alpha, interferon beta (fibroblast interferon), and interferon gamma (fibroblast interferon). Examples of such cytokines include, but are not limited to, erythropoietin (epoetin), granulocyte CSF (filgrastim), and granulocyte macrophage CSF (sargramostim). Immunomodulating agents other than cytokines include, but are not limited to, Calmett-Guerin Bacillus, Levamisole, and Ostreotide.

Examples of monoclonal antibodies against tumor antigens that can be used in combination with the lyophilized pharmaceutical composition of the invention include, but are not limited to, HERCEPTIN (R) (trastuzumab), RITUXAN (R) (Rituximab), MILOTARG (R) (anti-CD33), and CEMPAS (R) (anti-CD52).

In a further embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with a cancer vaccine, for example, a cancer vaccine selected from a cancer-testicular antigen (CTA) vaccine, such as a (CTA) vaccine, selected from NY-ESO-1, LAGE-1, MAGE-A1, -A2, -A3, -A4, -A6, -A10, -A12, CT7, CT10, GAGE1-6, GAGE 1-2, BAGE, SSX1 -5, SSX 2, HAGE, PRAME, RAGE-1, XAGE-1, MUC2, MUC5B and HMW-MAA. Non-limiting examples of CTA-based vaccines include MAGE-A3 vaccines (e.g. recMAGE-A3), NY-ESO-1, and PRAME.

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with a T-cell activator, for example, a T-cell activator, which is an antibody (optionally MAT), for example, selected from (a) a CD137 agonist; (b) a CD40 agonist; (c) an OX40 agonist; (d) MAT to PD-1; (e) MAT to PD-L1; (f) MAT to CTLA-4; and (d) combinations (a) - (f). In some embodiments, the additional therapeutic component is trelimumumab or imilimumab.

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with carboplatin to treat platinum drug-resistant recurrent ovarian cancer.

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used to treat hepatic cell carcinoma (for example, after sorafenib is ineffective).

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with irinotecan to treat metastatic colon cancer.

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with 5-fluorouracil (5-FU), leucovorin, oxaliplatin for the treatment of metastatic colon cancer.

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with cytarabine and fludarabine to treat childhood re / refractory AML.

In another embodiment, the lyophilized pharmaceutical composition of the invention can be used in combination with a JAK2 inhibitor to treat myoproliferative neoplasia.

The lyophilized pharmaceutical composition of the invention and any other therapeutic agents can be presented separately or presented together in a pharmaceutical package, kit or package for a patient.

The lyophilized pharmaceutical composition of the invention and combinations with other therapeutic agents or radiation therapy as described above can be administered for a long time to maintain beneficial therapeutic effects, or can be administered only for a short period. Alternatively, they can be administered in a pulsating or continuous manner.

The lyophilized pharmaceutical composition of the invention can be administered in an effective amount, i.e. in an amount that is effective to obtain the desired therapeutic effect, either individually (in monotherapy) or in combination with one or more chemotherapeutic agents or radiation therapy. For example, an “effective amount” may be an amount of a compound which, when administered to an individual suffering from a malignant tumor, slows the growth of the tumor, alleviates the symptoms of the disease, and / or increases the life expectancy.

The amount of lyophilized pharmaceutical composition of the invention administered to an individual may depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, gender, body weight and drug tolerance. The person skilled in the art is able to determine the appropriate dose depending on these and other factors. Effective doses for commonly used antitumor drugs and radiation therapy are well known in the art.

The purity of the compounds of the invention

Any compound herein can be purified. The compound herein may be at least 1% purity, at least 2% purity, at least 3% purity, at least 4% purity, at least 5% purity, at least 6% purity of at least 7% purity, at least 8% purity, at least 9% purity, at least 10% purity, at least 11% purity, at least 12% purity, at least 13% purity, at least 14% purity, at least 15% purity, at least 16% purity, at least 17% purity, at least 18% purity, at least at least 19% purity, at least 20% purity, at least 21% purity, at least 22% purity, at least 23% purity, at least 24% purity, at least 25% purity, at least 26% purity, at least 27% purity, at least 28% purity, at least 29% purity, at least 30% purity, at least 31% purity of at least 32% purity of at least re, 33% purity, at least 34% purity, at least 35% purity, at least 36% purity, at least 37% purity, at least 38% purity, at least 39% purity, at least 40% purity, at least 41% purity, at least 42% purity, at least 43% purity, at least 44% purity, at least 45% purity of at least 46% purity, at least 47% purity, at least 48% purity, at least 49% purity, at least 50% purity, at least 51% purity, at least 52% purity, at least 53% purity, at least 54% purity, at least 55% purity, at least 56% purity, at least 57% purity, at least at least 58% purity, at least 59% purity, at least 60% purity, at least 61% purity, at least 62% purity, at least 63% purity, at least 64% purity, at least 65% purity, at least 66% chi heights of at least 67% purity, at least 68% purity, at least 69% purity, at least 70% purity, at least 71% purity, at least 72% purity, at least 73% purity, at least 74% purity, at least 75% purity, at least 76% purity, at least 77% purity, at least 78% purity, at least at least 79% purity, at least 80% purity, at least 81% purity, at least 82% purity, at least 83% purity, at least 84% purity, at least 85% purity, at least 86% purity, at least 87% purity, at least 88% purity, at least 89% purity, at least 90% purity, at least 91% purity of at least 92% purity, at least 93% purity, at least 94% purity, at least 95% purity, at least 96% purity, at least 97% purity, at least 98% purity, at least 99% purity, at at least 99.1% purity, at least 99.2% purity, at least 99.3% purity, at least 99.4% purity, at least 99.5% purity, according to at least 99.6% purity, at least 99.7% purity, at least 99.8% purity, or at least 99.9% purity.

Examples

EXAMPLE 1. Obtaining a lyophilized composition of the sodium salt of the compounds of formula (1).

The sodium salt of the compound of formula (1) was dissolved in DMSO at a certain concentration using a vertical stirrer in a stainless steel vessel (HC) of the appropriate size. After the drug was completely dissolved in DMSO, samples from the bulk of the solution were tested using the UV or HPLC method without stopping the process to establish that the amount of the sodium salt of the compound of formula 1 was in the range of 95-105% of the target concentration. The bulk of the solution was filtered through a series of two pre-sterilized 0.2-micron sterilizing filters that were compatible with DMSO and collected in a two-liter buffer tank from HC. The filtration rate was constantly adjusted by visual observation of the amount available to fill the buffer tank. Then aliquots of one gram from the filtered main volume of the solution were filled depyrogenated transparent glass vials of 5 cm ³ . Each bottle was automatically partially closed on the filling line with a fluoropolymer-coated chlorobutyl rubber stopper for lyophilization, which was previously sterilized. Product vials were transferred to a lyophilizer under aseptic transfer conditions to initiate a lyophilization cycle. The lyophilizer used was a semi-industrial lyophilizer, Lyobeta 35, IMA-Telstar, which had a chamber volume of 1.02 m ² , an ice capacity of 35 kg, 22 kg / 24 hours — a condenser capacity.

Vials containing the solution were lyophilized using the cycle parameters shown in Table 1 below.

TABLE 1 - Operating parameters of the lyophilization cycle Stage Event Temperature / Pressure / Time T (° C) P Time (h) Loading five Atmospheric 0,0 First stage of freezing Temperature change -45 Atmospheric 1,0 First stage of freezing Temperature maintenance -45 Atmospheric 1,5 First stage of heating Temperature change 0 Atmospheric 1.3 First stage of heating Temperature maintenance 0 Atmospheric 2.0 The second stage of freezing Temperature change -45 Atmospheric 2.0 The second stage of freezing Temperature maintenance -45 Atmospheric 2.0 Primary drying step Pressure reduction and maintenance -45 6 mbar 4.0 Primary drying step Temperature change -20 6 mbar 3.0 Primary drying step Temperature maintenance -20 6 mbar 12.0 Primary drying step Temperature change -five 6 mbar 3.0 Primary drying step Temperature maintenance -five 6 mbar 24.0 The stage of secondary drying Temperature change 65 6 mbar 6.0 The stage of secondary drying Temperature maintenance 65 6 mbar 15.0

After completion of the lyophilization cycle, the lyophilizer was filled with nitrogen, and the vials were automatically completely closed. The bottles were aseptically transferred to the insulator, where each bottle was automatically closed with a blue aluminum stopper with a tear-off pad. Vials were checked visually before proceeding to sampling for control at the time of product release, and for labeling and packaging. Vials were stored at 2-8 ° C until ready. Each vial was labeled by its contents.

EXAMPLE 2. Comparative tests

I. Lyophilized formulations produced by the method of the invention:

Stock solutions were prepared containing the sodium salt of the compound of formula (1) in four different concentrations in DMSO, and the resulting solutions (labeled A to D) were filled into lyophilization bottles and lyophilized using the protocol described in Example 1 above. Pirani sensors and Baratron was used to determine the end of the stage of primary drying (sublimation). Figure 1 shows a progressive decrease in the content of DMSO over time during the stages of primary and secondary drying.

After lyophilization, lyophilized samples were analyzed for purity (% purity by HPLC), residual DMSO and residual moisture. Samples were reconstituted by dissolving them in the non-aqueous solvent system described in TABLE 2 below, and the recovery time and appearance of the reconstituted compositions were analyzed.

TABLE 2 - Recovery solvent % of each ingredient Purity Function Propylene glycol 65 National Formulary (NF), European Pharmacopoeia (PhEur) Solvent Glycerol 25 NF, PhEur Solvent Alcohol / Ethanol ten United States Pharmacopeia (USP), PhEur Thinning agent

The analysis results are presented in table 3 below.

Results for four different concentrations, n = 1

TABLE 3 Sample id A (100 mg / ml) B (75 mg / ml) C (50 mg / ml) D (25 mg / ml) Analysis % purity by HPLC (API purity 93.6%) 93.2 93.1 93.2 93.2 % residual solvent DMSO 19,4 15.1 19.2 20.8 Residual moisture <LOQ <LOQ <LOQ <LOQ Recovery Time (Manual) 17 min 40 sec 12 min 51 sec 12 min 49 sec 18 min 51 sec Appearance of reconstituted solution Transparent, slightly yellowish solution

LOQ = limit of quantification

II. Compositions for comparison:

Stock solutions of the sodium salt of the compound of formula (1) with a concentration of 100 mg / ml were lyophilized using the apparatus described in Example 1 above, but with a different temperature profile, which did not include the first stage of heating during freezing of the solution, but included freezing of the composition at different rates freezing.

The characteristics of the compositions for comparison, prepared in this way are shown below in table 4 .

Table 4 Identification number FP1 FP2 FP3 Analysis Characteristic results The appearance of sediment (all vials) Description Compact sediment separated from the walls Compact sediment separated from the walls Dense cracked sediment attached to the bottom of the vial Appearance of the reconstituted solution and recovery time Particle-free, clear solution Clear solution with particles adhering to the walls
> 30 minutes for complete dissolution Clear solution with particles adhering to the walls
> 30 minutes for complete dissolution Clear solution with particles adhering to the walls
> 30 minutes for complete dissolution Water content below 1% (hypothetically) 0.02% 0.005% 0.001% Residual DMSO Solvent Results Report for Information 19.1% (FP1-9) 19.4% (FP2-9) 19.4% (FP3-9)

III. Comparison of the results obtained for the compositions described in I and II

The results shown in step I above demonstrate that when the intermediate heating step (the "first heating step") is turned on during the freezing of the solution before primary drying in accordance with the invention, the result is a lyophilized dry composition that can be recovered in less than 20 minutes, and less than 15 minutes in some cases.

For comparison, the comparative compositions FP1, FP2 and FP3 described in II above and prepared by a process in which there is no intermediate heating step took longer to recover (more than 30 minutes). Not wishing to be bound by any theory, we believe that the intermediate stage of heating has the effect of increasing the porosity of the lyophilized product and increasing the surface area available for contact with solvent molecules, thereby increasing the solubility of the compositions.

IV. Comparison of drying time with Example 4 of WO2013 / 033176

Example 4 in WO2013 / 033176 describes the lyophilization of a sodium salt solution of a compound of formula (1) using the cycle parameters shown in Table 5 below.

Table 5 Stage Event Temperature / Pressure / Time T (° C) P Time (minutes) Stage of freezing Temperature change -40 Atmospheric 133 Stage of freezing Temperature maintenance -40 Atmospheric 360 Primary drying step Change in temperature and pressure -five 100 mTorr 117 Primary drying step Maintaining temperature and pressure -five 100 mTorr 1440 Primary drying step Temperature change ten 100 mTorr 50 Primary drying step Temperature maintenance ten 100 mTorr 1440 The stage of secondary drying Change in temperature and pressure thirty 50 mTorr 67 The stage of secondary drying Maintaining temperature and pressure thirty 50 mTorr 1440 The stage of secondary drying Temperature change 60 50 mTorr one hundred The stage of secondary drying Temperature maintenance 60 50 mTorr 1440 Total lyophilization time 6587 minutes = 109 hours and 47 minutes

The total lyophilization time for the composition described in Example 4 of WO2013 / 033176 is 109 hours and 47 minutes. For comparison, the total lyophilization time for the composition according to the invention, as described above in I, was 76.8 hours, i.e. more than 30 hours shorter than the total lyophilization time, for example, for 4 in WO2013 / 033176. Most of the differences are due to the significantly reduced stage of secondary drying in the method of the present invention compared to the method described in example 4 of WO2013 / 033176 (21 hours versus 50.78 hours). In the method of the present invention, the intermediate heating step (first) is located between the two freezing steps when the solution is initially frozen, and it is believed that this leads to a much more porous structure from which DMSO can more easily sublimate in the primary drying step. Thus, most of the DMSO is removed during the primary drying step, whereby a shorter secondary drying step can be used.

Thus, to summarize, the method of the present invention can reduce the time required to obtain a lyophilized product, which greatly improved the dissolution characteristics.

EXAMPLE 3. Large-scale studies on formulations A and B at 75 mg / ml and 100 mg / ml.

The results obtained in the experiments described in example 2 showed that the lowest levels of residual DMSO were obtained with composition B, in which the main solution containing the active compound at a concentration of 75 mg / ml was lyophilized. Confirmation studies were thus carried out on solutions with a concentration of 75 mg / ml and 100 mg / ml sodium salt of the compounds of formula (1) in DMSO. Lyophilization was carried out in the format of 100 vials, and analysis was performed on several samples. Used the protocol described in example 1. The properties of the obtained lyophilized products are shown below in table 6 .

TABLE 6 Sample id
Analysis 100 mg / ml 75 mg / ml % residual DMSO wt./mass., n = 3 17.4 (24.2 mg / vial) 18.7% (25.4 mg / vial) Recovery time (minutes), n = 3 8 minutes * 8 minutes * Appearance, n = 3 Transparent and colorless ** Water content, n = 2 <LOQ <LOQ Analyzed% wt./mass., N = 2 107.8 105

* Recovery time does not include diffusion of bubbles, which may take approximately an additional 10 minutes. However, the recovery was carried out manually, and it did not require a mechanical mixing apparatus.

** Although not found in this case, there may be cases where the solutions are slightly foggy and / or slightly white to yellow in color.

The results in Table 6 demonstrate that the method of the invention can be used to produce lyophilized formulations that have a dissolution time of less than ten minutes (except for the time required to clear the bubbles) and that the reduction can be carried out manually without the need for mechanical stirrers.

EXAMPLE 4. Obtaining the sodium salt of the compounds of formula (1).

The sodium salt of the compound of formula (1) was prepared as described in US 7,700,567 (the contents of which are hereby incorporated by reference) by compound 1c (where R ₁ = carbamate protecting group) with a phosphoramidite element of structure 1d:

A solid glass substrate with a predetermined pore size (CPG) of 1c coupled to protected 2'-deoxyguanosine (where R ₁ = tert-butyl phenoxyacetyl) was coupled to 2-2.5 equivalents of phenoxyacetyl decitabine phosphoramidite ( 1d , where R ₁ = phenoxyacetyl) in the presence of 60% of 0.3 M benzylthiotetrazole activator (in acetonitrile) for 10 minutes. A solid CPG support containing a protected DpG dinucleotide was treated with 20 ml of 50 mM K ₂ CO ₃ in methanol for 1 hour and 20 minutes. The bound product was oxidized, the protective group was removed, and the resulting compound was washed, filtered and purified by HPLC ÄKTA Explorer 100 with a preparative Gemini C18 column (Phenomenex), 250 × 21.2 mm, 10 μm with a protective column (Phenomenex), 50 × 21 , 2 mm, 10 μm, with 50 mM triethylammonium acetate (pH 7) in MilliQ water (mobile phase A) and 80% acetonitrile in MilliQ water (mobile phase B), from 2% to 20/25% of mobile phase B in volumes columns.

Negative Spray Ionization Mass Spectrometry (ESI-MS (-ve) DpG dinucleotide 2b :

where X + = triethylammonium (the calculated exact mass for the neutral compound C ₁₈ H ₂₄ N ₉ O ₁₀ P is 557.14), showed a mass / charge of 556.1 [MH] ^- and 1113.1 for [2M-H] ^- (cm mass spectrum of FIG. 31 in US 7,700,567).

The sodium salt of the compound of formula (1), i.e. DpG dinucleotide 2b , where X + = sodium, was obtained by re-dissolving the triethylammonium salt in 4 ml of water, 0.2 ml of a 2M NaClO ₄ solution. When 36 ml of acetone was added, the dinucleotide precipitated. The solution was stored at -20 ° C for several hours and centrifuged at 4000 rpm. for 20 minutes. The supernatant was removed and the solid precipitate was washed with 30 ml of acetone, followed by further centrifugation at 4000 rpm. for 20 minutes. The precipitate, which was dissolved in water and lyophilized, showed a mass / charge of 556.0 [MH] ^- (see mass spectrum in figure 36 of US 7700567).

OPTIONS FOR CARRYING OUT

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1. A method for producing a lyophilized pharmaceutical composition, comprising dissolving a compound of formula (1):

(1),

(1),

or a pharmaceutically acceptable salt thereof in a solvent containing dimethyl sulfoxide (DMSO) to form a solution, where the solvent is then removed by freeze drying to obtain a freeze dried product, where the freeze drying method comprises: (i) a first freezing step in which the solution is frozen, lowering its temperature to a temperature of not more than approximately -20 ° C; (ii) a first heating step in which the temperature of the frozen solution is induced to a temperature in the range of about -15 ° C to about 5 ° C, where the temperature in the range of about -15 ° C to about 5 ° C keeps the solution frozen; (iii) a second freezing step in which the temperature of the solution is lowered to a temperature not greater than about −20 ° C; (iv) a primary drying step, wherein the primary drying step comprises a sublimation step in which DMSO is removed from the solution in a frozen state by sublimation under reduced pressure to obtain a partially dried product; and (v) a secondary drying step in which DMSO is removed by evaporation from a partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product.

Embodiment 2. The method of Embodiment 1, wherein the compound of formula (1) is in the form of a sodium salt.

Embodiment 3. The method according to any one of Embodiments 1-2, wherein the solvent is non-aqueous.

Embodiment 4. The method of any one of Embodiments 1-3, wherein the lyophilized pharmaceutical composition has a dissolution time at ambient temperature and without mechanical stirring in a non-aqueous solvent containing 65% (v / v) propylene glycol, 25% (v / v) ./vol.) of glycerol and 10% (vol./about.) ethanol, not more than approximately 20 minutes.

Embodiment 5. The method according to any one of Embodiments 1-4, wherein in the amount of the lyophilized pharmaceutical composition obtained from 1 gram of solution, the content of residual DMSO is not more than about 20 mg.

Embodiment 6. The method of any one of Embodiments 1-5, wherein any residual DMSO is present in the lyophilized pharmaceutical composition in an amount corresponding to not more than 35 mg per 100 mg equivalent of the free base of the compound of formula (1).

Embodiment 7. The method of any one of Embodiments 1-6, further comprising packaging a lyophilized pharmaceutical in a sealed pharmaceutical container.

Embodiment 8. The method of any one of Embodiments 1-7, further comprising dissolving the lyophilized pharmaceutical composition in a solvent to form a liquid composition for injection.

Embodiment 9. The method of Embodiment 8 wherein the solvent is a non-aqueous solvent.

Embodiment 10. The method of any one of Embodiments 1-9, wherein the solution further comprises a co-solvent.

Embodiment 11. The method of Embodiment 1, further comprising reconstituting the lyophilized pharmaceutical composition in a pharmaceutically acceptable solvent to provide a liquid composition containing a compound of formula (1) or a pharmaceutically acceptable salt thereof.

Embodiment 12. The method of any one of Embodiments 1-11, wherein the reduced pressure in the primary drying step is from about 5 μbar to about 40 μbar.

Embodiment 13. The method of any one of Embodiments 1-12, wherein the temperature in the primary drying step is from about -3 ° C to about -9 ° C.

Embodiment 14. The method of any one of Embodiments 1-13, wherein the temperature in the secondary drying step is from about 30 ° C to about 65 ° C.

Embodiment 15. A pharmaceutical composition obtained by a method comprising the steps of: dissolving a compound of formula (1):

(1),

(1),

or a pharmaceutically acceptable salt thereof in a solvent containing dimethyl sulfoxide (DMSO) to form a solution, where the solvent is then removed by freeze drying to obtain a freeze dried product, where the freeze drying method comprises: (i) a first freezing step in which the solution is frozen, lowering its temperature to a temperature of not more than approximately -20 ° C; (ii) a first heating step in which the temperature of the frozen solution is induced to a temperature in the range of about -15 ° C to about 5 ° C, where the temperature in the range of about -15 ° C to about 5 ° C keeps the solution frozen; (iii) a second freezing step in which the temperature of the solution is lowered to a temperature not greater than about −20 ° C; (iv) a primary drying step, wherein the primary drying step comprises a sublimation step in which DMSO is removed from the solution in a frozen state by sublimation under reduced pressure to obtain a partially dried product; and (v) a secondary drying step in which DMSO is removed by evaporation from a partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product.

Embodiment 16. The pharmaceutical composition of Embodiment 15 wherein the compound of formula (1) is in the form of a sodium salt.

Embodiment 17. The pharmaceutical composition of any one of Embodiments 15-16, wherein the solvent is non-aqueous.

Embodiment 18. The pharmaceutical composition of any one of Embodiments 15-17, wherein the lyophilized pharmaceutical composition has a dissolution time at ambient temperature and without mechanical stirring in a non-aqueous solvent containing 65% (v / v) propylene glycol, 25% ( vol./about.) glycerol and 10% (vol./about.) ethanol, not more than approximately 20 minutes.

Embodiment 19. The pharmaceutical composition of any one of Embodiments 15-18, wherein in the amount of the lyophilized pharmaceutical composition obtained from 1 gram of solution, the content of residual DMSO is not more than about 20 mg.

Embodiment 20. The pharmaceutical composition of any one of Embodiments 15-19, wherein any residual DMSO is present in the lyophilized pharmaceutical composition in an amount corresponding to not more than 35 mg per 100 mg equivalent of the free base of the compound of formula (1).

Embodiment 21. The pharmaceutical composition of any one of Embodiments 15-20, wherein the method further comprises packaging the lyophilized pharmaceutical agent in a sealed pharmaceutical container.

Embodiment 22. The pharmaceutical composition of any one of Embodiments 15-21, wherein the method further comprises dissolving the lyophilized pharmaceutical composition in a solvent to form a liquid composition for injection.

Embodiment 23. The pharmaceutical composition of Embodiment 22 wherein the solvent is a non-aqueous solvent.

Embodiment 24. The pharmaceutical composition of any one of Embodiments 15-23, wherein the solution further comprises a co-solvent.

Embodiment 25. The pharmaceutical composition of Embodiment 15 wherein the method further comprises reconstituting the lyophilized pharmaceutical composition in a pharmaceutically acceptable solvent to provide a liquid composition containing a compound of formula (1) or a pharmaceutically acceptable salt thereof.

Embodiment 26. The pharmaceutical composition of any one of Embodiments 15-25, wherein the reduced pressure in the primary drying step is from about 5 μbar to about 40 μbar.

Embodiment 27. The pharmaceutical composition of any one of Embodiments 15-26, wherein the temperature in the primary drying step is from about −3 ° C. to about −9 ° C.

Embodiment 28. The pharmaceutical composition of any one of Embodiments 15-27, wherein the temperature in the secondary drying step is from about 30 ° C to about 65 ° C.

Claims (25)

1. A method of obtaining a lyophilized pharmaceutical composition, comprising dissolving a compound of the formula:

further indicated as a compound of formula (1), or a pharmaceutically acceptable salt thereof in a solvent containing dimethyl sulfoxide (DMSO) to form a solution, where the solvent is then removed by freeze drying to obtain a freeze dried product, where the freeze drying method comprises: (i) the first stage of freezing, in which the solution is frozen, lowering its temperature to a temperature not exceeding -20 ° C; (ii) a first heating step in which the temperature of the frozen solution is induced to a temperature in the range of -15 ° C to 5 ° C, where a temperature in the range of -15 ° C to 5 ° C keeps the solution frozen; (iii) the second stage of freezing, in which the temperature of the solution is lowered to a temperature not greater than -20 ° C; (iv) a primary drying step, wherein the primary drying step comprises a sublimation step in which DMSO is removed from the solution in a frozen state by sublimation under reduced pressure to obtain a partially dried product; and (v) a secondary drying step in which DMSO is removed by evaporation from a partially dried product in an unfrozen state under reduced pressure to obtain a lyophilized product. 2. The method of claim 1, wherein the compound of formula (1) is in the form of a sodium salt. 3. The method of claim 1, wherein the solvent is non-aqueous. 4. The method of claim 1, wherein the lyophilized pharmaceutical composition has a dissolution time at ambient temperature and without mechanical stirring in a non-aqueous solvent containing 65% (v / v) propylene glycol, 25% (v / v) glycerol and 10% (v / v) ethanol, not more than 20 minutes, where the lyophilized pharmaceutical composition contains 100 mg of the free base equivalent of the compound of formula (1) and where no more than 35 mg of residual DMSO is present in the composition. 5. The method according to p. 1, where in the amount of lyophilized pharmaceutical composition obtained from 1 gram of solution, the content of residual DMSO is not more than 20 mg. 6. The method according to p. 1, where any residual DMSO is present in the lyophilized pharmaceutical composition in an amount corresponding to not more than 35 mg per 100 mg equivalent of the free base of the compound of formula (1). 7. The method of claim 1, further comprising packaging the lyophilized pharmaceutical composition in a sealed pharmaceutical container. 8. The method according to p. 1, further comprising dissolving the lyophilized pharmaceutical composition in a solvent to obtain a liquid composition for injection. 9. The method of claim 8, wherein the solvent is a non-aqueous solvent. 10. The method of claim 1, wherein the solution further comprises a co-solvent. 11. The method according to claim 1, further comprising restoring the lyophilized pharmaceutical composition in a pharmaceutically acceptable solvent to obtain a liquid composition containing a compound of formula (1) or a pharmaceutically acceptable salt thereof. 12. The method of claim 1, wherein the reduced pressure in the primary drying step is from 0.5 Pa (5 μbar) to 4 Pa (40 μbar). 13. The method according to p. 1, where the temperature at the stage of primary drying is from -3 ° C to -9 ° C. 14. The method according to p. 1, where the temperature at the stage of secondary drying is from 30 ° C to 65 ° C. 15. Lyophilized pharmaceutical composition containing the compounds of formula (1):

(1) or its pharmaceutically acceptable salt obtained by the method according to any one of paragraphs. 1-14.

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